Observational Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Oncol. Sep 24, 2019; 10(9): 307-317
Published online Sep 24, 2019. doi: 10.5306/wjco.v10.i9.307
DNA extraction from paraffin embedded colorectal carcinoma samples: A comparison study of manual vs automated methods, using four commercially kits
Zsolt Kovacs, Ioan Jung, Erzsebet Csernak, Zoltan Szentirmay, Laura Banias, Genoveva Rigmanyi, Simona Gurzu
Zsolt Kovacs, Ioan Jung, Laura Banias, Simona Gurzu, Department of Pathology, University of Medicine, Pharmacy, Sciences and Technology, Targu-Mures 530149, Romania
Genoveva Rigmanyi, Simona Gurzu, Research Center (CCAMF), University of Medicine, Pharmacy, Sciences and Technology, Targu-Mures 540139, Romania
Author contributions: Kovacs Z drafted the article and contributed to the DNA purifications; Jung I contributed to the diagnosis and immunohistochemical assessment; Csernak E contributed to automated and manual DNA isolation; Szentirmay Z contributed to the research design; Banias Laura performed DNA collection; Rigmanyi G performed manual DNA extraction; Gurzu S designed research and confer the final agreement for publication; Zsolt Kovacs and Laura Banias have equally contribution to the paper.
Supported by the University of Medicine, Pharmacy, Science and Technology Research Grant, No. 275/11.01.2017.
Institutional review board statement: The agreement of the Ethical Committee of University of Medicine and Pharmacy, Targu-Mures, Romania, was obtained.
Informed consent statement: This is a retrospective study. No consent was necessary.
Conflict-of-interest statement: The authors have declared no conflicts of interest.
Data sharing statement: No additional data are available.
STROBE statement: The authors have read the STROBE Statement-checklist of items, and the manuscript was prepared and revised according to the STROBE statement-checklist of items.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Simona Gurzu, MD, PhD, Chief Doctor, Research Center (CCAMF), University of Medicine, Pharmacy, Sciences and Technology, 38 Gheorghe Marinescu Street, Targu-Mures 540139, Romania. simonagurzu@yahoo.com
Telephone: +40-745-673550 Fax: +40-265-210407
Received: March 12, 2019
Peer-review started: March 20, 2019
First decision: April 15, 2019
Revised: August 19, 2019
Accepted: September 4, 2019
Article in press: September 5, 2019
Published online: September 24, 2019
Processing time: 203 Days and 9.3 Hours
Abstract
BACKGROUND

Nucleic acid isolation from formalin-fixed, paraffin-embedded tissue (FFPET) samples is a daily routine in molecular pathology laboratories, but extraction from FFPET is not always easily achieved. Choosing the right extraction technique is key for further examinations.

AIM

To compare the performance of four commercially available kits used for DNA extraction in routine practice.

METHODS

DNA isolation was performed on 46 randomly selected formalin-fixed, paraffin-embedded (FFPE) colorectal adenocarcinoma (CRC) surgical specimens. Four commercially available extraction kits were used: two for manual DNA extraction (the PureLink Genomic DNA Mini Kit from Invitrogen and the High Pure FFPE DNA Isolation Kit from Roche) and two for automated DNA extraction (the iPrep Genomic DNA Kit from Invitrogen and the MagnaPure LC DNA Isolation Kit from Roche). The DNA concentration and quality (odds ratio) among the four systems were compared. The results were correlated with the clinicopathological aspects of CRC cases: age, gender, localization, macro- and microscopic features, lymph node metastases, and the lymph node ratio.

RESULTS

The highest DNA concentration was obtained using the manual kits: 157.24 ± 62.99 ng/µL for the PureLink Genomic DNA Mini Kit and 86.64 ng/µL ± 43.84 for the High Pure FFPE DNA Isolation Kit (P < 0.0001). Lower concentrations were obtained with automated systems: 20.39 ± 21.19 ng/µL for the MagnaPure LC DNA Isolation Kit and 8.722 ± 6.408 ng/µL for the iPrep Genomic DNA Kit, with differences between the systems used (P < 0.0001). The comparison between age, gender, tumor localization, pT or pN stage and the lymph node ratio indicated no statistically significant difference in DNA concentration using any of the nucleic acid isolation kits. DNA concentration was influenced by the macroscopic features and grade of differentiation. A higher DNA concentration was obtained for well-differentiated polypoid colorectal adenocarcinomas (CRCs), compared with undifferentiated ulcero-infiltrative carcinomas, irrespective of the kit used.

CONCLUSION

For research or diagnosis that needs high DNA concentrations, manual methods of DNA isolation should be used. A higher amount of DNA can be obtained from polypoid-type differentiated CRCs. Automated systems confer comfort and a lower amount of DNA that is, however, sufficient for classic polymerase chain reaction (PCR) and real-time quantitative PCR molecular examinations. All four commercially available kits can be successfully used in daily practice.

Keywords: DNA isolation; Colorectal cancer; Paraffin-embedded; PureLink Genomic DNA Mini Kit; High Pure FFPE DNA Isolation Kit; iPrep Genomic DNA Kit; MagnaPure LC DNA Isolation Kit

Core tip: In this paper we proved the advantages and disadvantages of manual vs automated methods of DNA extraction. The original aspect refers to the correlation between DNA concentration and colorectal adenocarcinoma (CRC) features. We conclude that manual methods are more suitable for obtaining high DNA concentrations, especially from differentiated polypoid-type CRCs. In CRC samples, a higher DNA concentration is associated with a lower OD value.